The goal of our investigations is the understanding of the molecular mechanisms that regulate cytoplasmic mRNA decay in eucaryotes. Altered control of mRNA stability can lead to aberrant gene regulation and, consequently to disease. Our studies have focused on the characterization of the cis-acting elements and trans-acting factors involved in mRNA turnover in the yeast Saccharomyces cerevisiae. Through the efforts of a number of laboratories it is now clear that poly(A) shortening and/or hydrolysis of the 5' cap structures for many transcripts are important rate determining events in controlling the stability of a given transcript in both mammalian and yeast cells. Based on these results, the goal of the experiments in this grant proposal is the identification and characterization of the genes and their products that regulate decapping activity in yeast. To accomplish this, a biochemical screen was developed to monitor decapping activity in cell extracts. Extracts prepared from a collection of temperature-sensitive strains were screened for reduced decapping activity. This analysis successfully identified factors that modulate decapping activity. The results demonstrated that proteins encoded by the VPS16, MRT1 and the heat shock protein SSA1/SSA2 appear to be regulators of the decapping activity, with Ssa1p/2p being a direct effector of the decapping enzyme. The ability of Ssa1p/2p to modulate decapping activity depends on whether ADP is present in the reaction. Furthermore, evidence is presented suggesting that the Dcp1p protein may be part of a degradosome complex consisting of, at a minimum, Dcp1p, Xrn1p, Vps16p, Mrt1p and the heat shock protein Ssa1p/2p. We have also identified a dcp2 allele that may encode an inducible decapping activity. Based on these results, the aims of the grant proposal are; 1) Characterization of the role cytosolic Hsp70p and its regulators in controlling mRNA turnover; 2). Biochemical characterization of the "degradosome complex"; C) Molecular, genetic and biochemical characterization of the individual factors that are associated with the degradosome complex.